Progressive supranuclear palsy (PSP) is the second most common Parkinsonian disorder with complex etiology. The underlying molecular mechanism of PSP pathogenesis remains unclear. The present study aims to find the feasibility of using plasma miRNAs as novel biomarkers. Plasma-focused qPCR panels were used for microRNA profiling and identified differentially expressed microRNAs in PSP compared to controls. The DIANA-miRPath v3.0 was used to perform KEGG pathway analysis. We then confirmed the expression of selected candidates by RT-qPCR and their clinical utility was assessed by ROC analysis. Profiling data revealed 28 differentially expressed microRNAs in PSP. Five overexpressed miRNAs were selected for further analysis. The KEGG pathway analysis revealed 48 high-risk pathways. The study revealed that as a single marker—miR-19b-3p, miR-33a-5p, miR-130b-3p, miR-136-3p, and miR-210-3p had a specificity of 64.71%, 82.35%, 68.75%, 82.35%, and 70.59% at sensitivity 77.78%, 77.78%, 66.67%, 73.33%, and 66.67%, respectively. The result suggests that circulating plasma miRNAs were altered in PSP compared to control. The findings of this study may provide potential biomarkers and pathways associated with PSP. Further large-scale validation studies are required to confirm the same. Progressive supranuclear palsy (PSP) is one of the most frequent atypical Parkinsonisms that affects 5–10/100,000 people, with no apparent variation across geographical locations, ethnicity, or race (aged > 60 years) . The majOver the past few years, there have been several attempts to understand the genetic basis of PSP and also to identify biomarkers that could aid in establishing the diagnosis and assessing the progression of PSP. Few studies have suggested the level of serum/plasma neurofilament light chain or tau forms as markers of diagnosis and disease severity ,7,8. FurA growing body of evidence suggests that microRNAs (miRNAs or miRs), a class of non-coding endogenous single-stranded, small (18–25 nucleotide) RNAs that regulate gene expression at the post-transcriptional level in many neurodegenerative diseases , have imIn this study, we examined differentially expressed plasma miRNAs of PSP compared to healthy participants. Subsequently, the functions, pathways, and common hub genes of candidate miRNAs were predicted by bioinformatics analysis. The feasibility of using plasma miRNAs as novel biomarkers for PSP was evaluated by quantitative real-time PCR and ROC analysis.In this prospective study, we randomly recruited 18 patients (aged 60.11 ± 1.6 years) with probable PSP within three years of the clinical diagnosis from the Outpatient Services of the Department of Neurology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, from July 2017 to March 2020. Movement Disorder Society diagnostic criteria [Fasting venous blood (5 mL) was collected by venepuncture from all study participants and transferred to EDTA vacutainers for further processing. The EDTA treated blood sample was centrifuged at 3000 rpm for 15 min at room temperature to separate blood cells from the liquid part of the blood called plasma. The separated plasma was collected carefully without disturbing the buffy coat and, again, the plasma sample was centrifuged to minimize buffy coat contamination and then aliquoted for further study. The aliquoted plasma sample was stored at −80 °C or used immediately for miRNA isolation. The entire process of collecting, separating, and storing plasma samples was completed within 45 min.MicroRNAs were extracted using the miRNeasy Serum/Plasma Advanced Kit (50) with spike-ins  as per the manufacturer’s instructions. While extracting the sample, 1 µg of carrier RNA was added  to each sample. The extracted miRNAs were then reverse transcribed using the miRCURY LNA RT Kit  in a total of 10 µL volume reactions containing 4 µL of isolated miRNAs as per the manufacturer’s instruction. For qPCR amplification, 3 µL of 20× diluted cDNA was assayed in a 10 µL PCR reaction using miRCURY LNA SYBR Green PCR master mix . The plasma samples of the validation cohort were processed similarly.https://dataanalysis2.qiagen.com/ accessed on 11 December 2019) and followed onscreen instructions to identify differentially expressed miRNAs.For the miRNA screening, miRCURY LNA miRNA Focus PCR Panels  were used. Each panel consists of pre-coated primers for 179 miRNAs, including cel-miR-39-3p, UniSp2, UniSp4, UniSp5, and UniSp6. In addition, each 96-well PCR panel contained an inter-plate calibrator in triplicate and an empty negative control. The miRNA profiling experiment consisted of pooled samples from PSP patients, as well as healthy subjects in triplicate . The qPChttp://mirdb.org accessed on 22 August 2021), TargetScan v7.2 (http://www.targetscan.org/vert_72/ accessed on 22 August 2021), and miRWalk  individually. Only genes recorded for each of the miRNAs in three databases were considered as ultimate target genes of that particular candidate miRNA, and they were mapped to the miRPath v3.0 tool (http://snf-515788.vm.okeanos.grnet.gr/ accessed on 23 August 2021) to identify risk pathways and associated genes [p-value of <0.05 was set as a threshold. Risk pathways regulated by at least two miRNAs were identified and enlisted.A number of computational methods for identifying miRNA target genes have already been developed. In our study, the target genes of all five candidate miRNAs were predicted using miRDB  was measured in triplicate using qPCR, with miR-16-5p serving as an endogenous reference. p < 0.05 being regarded as statistically significant. The Holm–Sidak technique was used to compensate for multiple comparisons. The clinical usefulness of candidate miRNAs was assessed using receiver operating characteristic (ROC) curves. Data are given as means with standard error (S.E).GraphPad Prism v.6.0 was used for statistical analysis . The nonparametric Mann–Whitney test was used to compare the data of two groups, with p < 0.05 with a fold change of <1.5 or >1.5) were enlisted  was performed to know the similarity between the miRNA expression patterns in PSP and control groups a, and thenlisted . The hieenlisted . We chosSynthetic spike-ins were utilized to test the quality of plasma miRNA isolation (UniSp2) and cDNA synthesis efficiency (UniSp6). The average threshold cycle (Ct) values of the spike-ins in all the samples were 20.02 and 18.92, respectively, with a low standard deviation of 0.02 and 0.06, indicating that the amount of miRNA in each sample examined in the qPCR was identical. Two miRNAs, miR-451a and miR-23a, were used to measure hemolysis in all samples. The Ct difference between these two miRNAs was <5, suggesting that the samples had not been hemolyzed and were therefore appropriate for further investigation.The expressions of selected candidate miRNAs were tested in 17 healthy controls and 18 PSP patients who were enrolled in the study individually. The findings indicated that putative candidate miRNAs were overexpressed in plasma samples of patients, and they complemented the initial screening data . The ROCp < 0.05, which are listed in Every miRNA can interact with multiple genes. The target genes of candidate miRNAs selected for validation were predicted and verified by miRDB, TargetScan, and miRWalk. The common target genes regulated by each candidate miRNA in all the databases were used for identifying pathological pathways associated with PSP. Then, we performed the KEGG pathway analysis for the candidate miRNAs to learn about the miRNA-mRNA network using the online tool—DIANA miRPath v3.0. This tool detected 48 KEGG pathways with The most prevalent form of atypical Parkinsonism is PSP, which is a rare neurological illness. Because the symptoms of PSP are similar to those of other movement disorders ,5, it isThe current study discovered changes in the expression of 28 miRNAs in PSP, with 23 being upregulated and 5 being downregulated. Five of the 23 increased miRNAs were chosen for further study: miR-210-3p, miR-19b-3p, miR-33a-5p, miR-130b-3p, and miR-136-3p. There have been few results of miRNA profiles in PSP, and these miRNAs have not been documented in prior PSP investigations. Previous studies on post-mortem brain tissues discovered that miR-132 was downregulated whereas miR-147a and miR-518e were upregulated ,16. A miIn this study, we describe a network of miRNAs—target genes—signaling pathways in PSP, which may aid in understanding the etiology of PSP and associated diseases. Our study revealed that the candidate miRNAs might be linked to a number of hub genes. Notably, when we compared the target genes of miR-132, miR-147a, and miR-518e with the Transforming growth factor (TGF)-β pathway and FoxO signaling pathways have been implicated in many cellular processes and modulated by at least three of the identified candidate miRNAs . Thus, tThe upregulation of miR-210-3p was the most notable discovery of the current investigation. It is worth noting that miR-210-3p can target several genes involved in cell division, migration, mitochondrial metabolism, angiogenesis, DNA repair, and chromatin remodeling, among other cellular processes . MiR-210E. coli-induced neuroinflammation by targeting TNFAIP3, a negative regulator of NF-kB activation [Another intriguing discovery was increased circulating levels of miR-19b-3p in PSP patients. Changes in this miRNA’s expression have previously been linked to a variety of neurodegenerative disorders, including tauopathies. The serum levels of miR-19b-3p were shown to be downregulated in PD patients with LRRK2A G2019S mutations , whereastivation . A recentivation . These dIn PSP patients, circulation levels of miR-33a-5p were also found to be considerably greater. The miR-33 family of miRNAs, which includes miR33a and miR-33b, has several target genes and is primarily engaged in modulating the expression of genes that regulate cholesterol and fatty acid production . NotablyPSP patients had considerably greater circulating levels of miR-130b-3p than controls. Multiple genes are targeted by miR-130b-3p. Certain tumors were found to have high levels of miR-130b-3p. Upregulation of miR-130b promotes the viability of prostate cancer cells while inhibiting apoptosis . PTEN haIn PSP patients, circulating levels of miR-136-3p were also increased. PTEN and Kruppel-like factor 7 (KLF7) are two of miR-136-3p’s target genes ,55. KLF7The miRNA databases employed for bioinformatics analysis are the study’s principal shortcoming. Because these databases may have restricted data sources, our study may need to be modified in the future as databases become more comprehensive and revised. Second, the present sample size of 35 participants is insufficient for a conclusion to be drawn. We chose sample pooling for initial miRNA screening because it decreased costs, shortened analytical run times, and compensated for the limited number of plasma samples available, allowing us to profile a significant number of miRNAs in both patient and control samples at a low cost. Pooling of biological specimens has been used in earlier studies to identify miRNA as a biomarker . AlthougThe most frequently used diagnostic approaches for PSP are neuroimaging and rating scales. PSP diagnosis based on these methods is challenging and limited due to their lack of usefulness in a wide clinical environment. To address this, we employed miRNA profiling and identified five plasma miRNAs that are overexpressed in PSP patients compared to healthy controls. At present, this miRNA panel has the potential to be used as a secondary diagnostic tool for detecting PSP in its early stages with more confidence. The identified miRNAs regulate a variety of important cellular processes and their target genes, all of which might be linked to PSP pathogenesis. However, large-scale research will have to validate this.


The versatile adsorption properties of this material were evaluated by quantum chemistry theory calculations and batch adsorption experiments using five nucleotides as probes. The quantum chemistry results demonstrated that CTF-DBF can participate in multiple interactions with nucleotides. All the analyses performed present good linearity with R2 > 0.9993. The detection limits of targets ranged from 0.3 to 0.5 mg/kg, the spiked recoveries were between 85.8 and 105.3% and the relative standard deviations  were between 1.1 and 4.5%. All these results suggest that this versatile CTF-DBF has great potential for sample pretreatment.The aromatic structure and the rich nitrogen content of polymers based on covalent triazine-based frameworks (CTF) and their unique hydrophilic-lipophilic-balanced adsorption properties make them promising candidates for an adsorbent that can be used for sample pretreatment. Herein, a new covalent triazine-based framework (CTF-DBF) synthesized by a Friedel–Crafts reaction was used for the determination of the content of nucleotides in commercial infant formula. It was shown that the synthetic materials had an amorphous microporous structure, a BET surface area of up to 595.59 m Nucleotides identified as conditionally essential nutrients are widely added to infant formula. Studies have shown that the addition of nucleotides to milk powder promotes the growth and maturation of the gastrointestinal tract of infants ,2. ConseThe unique properties of covalent organic frameworks (COF), including their high surface area, physicochemical stability and structural diversity, have led to their receiving extensive attention during the past few years ,7,8,9,10As alternatives to COFs, covalent triazine-based organic framework/-polymers (CTF/CTP) comprised of alternating triazine-benzene units can be synthesized economically under relatively mild conditions ,24. The 13C NMR, nitrogen sorption analysis and thermogravimetric analysis. Nucleotides are compounds with a nitrogenous base and an aldopentose and at least one phosphate group, and triazines are aromatic heterocyclic rings containing N atoms and a π-electron structure. Accordingly, the proposed CTF-DBF has several possible interactions with nucleotides, including hydrogen bonding, hydrophobic interactions, electrostatic attraction, and π-π interaction and, as a result, could be an efficient solid phase-extraction (SPE) adsorbent for nucleotides. Five nucleotides were selected as probes to evaluate the adsorption capacity and mechanism of CTF-DBF. Quantum chemistry theory calculations were conducted to describe quantitatively the versatile adsorption properties of CTF-DBF. In addition, adsorption experiments were conducted to identify five nucleotides in the infant formula. The main factors that influence the SPE efficiency, such as the amount of adsorbent, composition, and volume of desorption solvent, times reused, and sample pH, were optimized, leading to a CTF-DBF with the potential to become a powerful food sample pretreatment adsorbent.Herein, we describe CTF-DBF, a new type of CTF synthesized according to the method proposed by Lars. This uses dibenzofuran (DBF) and cyanuric chloride  as monomers. The morphology and physicochemical properties of CTF-DBF were characterized by SEM, elemental analysis, FT-IR spectra, solid-state Aluminum trichloride, cyanuric chloride and dibenzofuran were purchased from Aladdin Reagent Co., Ltd. . Analytical grade 1,2-dichloroethane, ethanol, tetrahydrofuran, methanol, acetone, and other solvents were provided by Sinopharm Chemical Reagent Co., Ltd. . Uridine 5′-monophosphate (UMP), cytidine 5′-monophosphate (CMP), adenosine 5′-monophosphate (AMP), inosine 5′-monophosphate (IMP), and guanosine 5′-monophosphate (GMP) standards   using a standard Bruker magic angle-spinning probe with 4 mm (outside diameter) zirconia rotors. Transmission electron microscopy (TEM) images were produced on a TalosF200S TEM . A Nicolet 6700 spectrometer  was used to record the Fourier-transform infrared (FT-IR) spectra. N2 adsorption/desorption isotherms were measured on an Autosorb-1 MP gas adsorption instrument . Elemental analysis was accomplished with a Flash EA 1112 elemental analyzer . X-ray diffraction (XRD) spectra were collected from an ARL X′TRA diffraction-meter using Cu Kα radiation . Ultrapure water use in this work was taken from a Milli-Q gradient ultrapure water system . The HPLC separations were performed on Agilent 1260  with an Ultimate XB-C18 column  and a UV detector.Scanning Electron Microscope (SEM) images were collected on an S-4300 SEM configured with an energy dispersive spectrometer system . Solid-state The synthesis was conducted using published methods with some modifications. Briefly, dibenzofuran (2.52 g) dissolved in 1,2-dichloroethane (100 mL) was added dropwise to a mixture of cyanuric chloride (1.84 g) and aluminum trichloride (4.80 g) and the mixture was refluxed under a nitrogen atmosphere for 24 h. After cooling to room temperature (rt), the product was washed thoroughly with ice water, tetrahydrofuran, and ethanol three times to obtain a crude product. Soxhlet extraction was conducted for 48 h with a mixture of tetrahydrofuran and methanol as the extraction liquid to further purify the obtained product. The final product was vacuum dried for 12 h at 60 °C to obtain CTF-DBF as a brown powder. The yield of polymer is up to 85%. A schematic of this method is shown in n), calculated from Equation (1) changes on the formation of a so-called CTF-DBF-analyte supramolecule: CTF-DBF-n is adsorption energy between CTF-DBF and the target molecule, ECTF-DBF is energy of CTF-DBF and En is the energy of the target. All calculations were performed using the Gaussian 09 program. To explore the adsorption mechanism of the CTF-DBF adsorbent and nucleotides, their optimized molecular structures and adsorption energies of the nucleotides were obtained using density functional theory (DFT). Molecular docking was conducted using the AutoDock Vina program to help obtain the rational preliminary estimates of complexes of various nucleotides with CTF-DBF. The lowest energy structure of each complex was selected for further optimization at the B3LYP/6-31G  level. Finally, molecular non-covalent interaction (NCI) analysis was conducted by using the Multiwfn program. The stabilization energy (△EInfant formula milk powder (5 g) was dissolved into ultrapure water (20 mL) at 50–60 °C and the solution was cooled to rt. The obtained solution was first ultrasonicated for 10 min, then the pH was adjusted to 4.0 with acetic acid. The solution was then diluted to 50 mL and filtered for use in the following experiments.2 and the residues were redissolved in the mobile phase (1 mL).SPE measurements were performed to verify the adsorption capacity of the prepared CTF-DBF adsorbent. Briefly, the above filtrate (5 mL) was loaded onto a self-made 80 mg/3 mL CTF-DBF solid-phase extraction cartridge which had been pretreated with methanol (3 mL) and water (3 mL). Then the nucleotides were eluted with a 20% methanol aqueous solution (6 mL). The eluent was evaporated to dryness at rt under a gentle stream of N2PO4 .An Agilent 1260 with a UV detector was used to perform all the analyses in the experiments. A unitary C18 column  was used as the chromatographic separation column. The injection volume was set at 20 μL, the column temperature at 30 °C, the detection wavelength at 254 nm, and the flow rate at 0.8 mL/min. The mobile phase was composed of an aqueous solution of MeOH/KH−1 in CTF-DBF is significantly reduced, indicating the completion of the Friedel–Crafts alkylation reaction shown in −1) of the furan unit and the strong C=N vibrations (1521 cm−1and 1365 cm−1) confirm the existence of the triazine core  model and the quenched solid density functional theory (QSDFT) method were conducted to investigate the specific surface area and the pore size distribution, respectively. The physisorption experiments reveal an intrinsic and permanent surface area of 595.59 m2/g and a sharp pore size distribution  was used to explore the thermal stability of the material, and the result is shown in 2 carbon atoms of the dibenzofuran moiety (284.6 eV), the carbon atoms in the triazine node (285.8 eV), and the carbon atoms of C-O-C (288.7 eV) of the oxygens in CTF-DBF. All the above results further confirmed that the polymer had been successfully prepared.To gain insight into the local chemical environment, X-ray photoelectron spectroscopy (XPS) was conducted. The low-resolution XPS spectra demonstrated the existence of C 1s, O 1s, and N 1s electrons a. The hin) changes (n using Equation (1). From the results of quantum chemistry calculation, it can be seen that all the adsorption energies between CTF-DBF and guest molecules are negative, which implies that these so-called supramolecules are stable. In addition, the adsorption energies of CMP and GMP are significantly higher than those of IMP and UMP, which is due to the existence of amino groups which significantly enhance the hydrogen bonding interaction between CTF-DBF and guest molecules. However, the △E of IMP is higher than that of the nitrogen-rich compound UMP and why the significantly different structures compounds  should have similar △E values. This may be the result of the anisotropic of the multiple forces such as inclusion interactions, π-π interactions, and hydrogen bonding interactions generated among CTF-DBF and detection targets. For example, the hydrogen bonding of UMP, CMP, and AMP increased with the increase of nitrogen content and amino group number, while their inclusion decreased with the increase of molecular size, which led to similar △E values.To describe the interactions between CTF-DBF and CMP, UMP, IMP, AMP, and GMP directly and quantifiably, the optimized complex with the supramolecular structures of analyte-CTF-DBF at the lowest energy was first obtained using quantum chemistry calculations in vacuo and DFT/B3LYP/6-31G  as the basis set . As the  changes  upon the2PO4− in nucleotides can insert into or approach the cavity of CTF-DBF, forming O-H…N hydrogen bonds with the nitrogen heterocycle of CTF-DBF. The benzene ring and partially protonated nitrogen can control the formation and assembly of the supramolecular structure of the complex by forming hydrogen bond interactions and π-π interactions, while the benzene ring, nitrogen heterocycle, and electron-donating amino groups promote the formation of CH...π interactions, CH…O, OH…N, NH...O hydrogen bonds, and LP...π interactions. NCI analysis intuitively clarified the excellent adsorption potential of CTF-DBF for nucleotides from the theoretical perspective.To further confirm this inference, a non-covalent interaction (NCI) was conducted as a theoretical research strategy to explore the detailed intermolecular interactions between CTF-DBF and nucleotides. As shown in v/v). For the choice of the elution volume, varying volumes (4 to 12 mL) of 5% MeOH were applied for solid-phase extraction progress. As shown in An effective and sensitive SPE-HPLC method was developed for nucleotides in infant formula using CTF-DBF as a solid-phase extraction (SPE) adsorbent for enrichment and purification. The main factors, including the amount of adsorbent, sample pH, composition and volume of desorption solvent and the extent of reuse, all affect the extraction efficiency. All these main factors were investigated and evaluated in the sample pretreatment step to obtain the best extraction efficiency using 5 mL of a 100 μg/mL nucleotides standard solution sample as the test probe. The amount of sorbent is closely correlated with the quantity of adsorbed analytes, so the experiments were first studied with the sorbent amount ranging from 40 mg to 120 mg. As can be seen in 2) were studied and are summarized in Method validation methods, including limit of quantification (LOQ), limit of detection (LOD), linear range, and correlation coefficient  has been synthesized by a one-pot method. Its morphology and physicochemical properties were measured by SEM, FT-IR spectra, solid-state


Qi et al. recently proposed a nomogram to reveal the prognostic value of peripheral blood inflammatory indexes (named Risk) and predict overall survival (OS) in limited-stage small cell lung cancer (LS-SCLC). However, it hasn’t undergone external application so far. This study aimed to verify the role of Risk as a prognostic variable of OS and apply the nomogram externally.We used a retrospective analysis of clinical data of 254 patients diagnosed as LS-SCLC in Shanxi Cancer Hospital from January 2015 to December 2018 to apply Qi’s nomogram externally. We also performed subgroup analysis to explore the predictive value of Risk. The model was evaluated in terms of discrimination (the area under the ROC curve (AUC ROC) and calibration .The prognosis of patients with low-Risk was significantly better than those with high-Risk in our cohort (p<0.01). The AUC of 1-, 2-, and 3-year OS was 0.644, 0.666, and 0.635, respectively. The calibration curve showed a nearly ideal calibration-slope of 1-, 2-, and 3-year OS (1.00 (0.41-1.59), 1.00 (0.54-1.46) and 1.00 (0.43-1.57), respectively).The external application of nomogram added Risk for predicting OS in LS-SCLC patients showed a moderate-to-good performance using a cohort with different case-mix characteristics. The external application confirmed the predictive value of Risk and the usefulness of the nomogram for the prediction of OS. Lung cancer remains the leading cause of cancer mortality in China and worldwide , 2. SmalP < 0.001). Add Risk to nomogram, a remarkable improvement in predictive accuracy was observed compared with clinical factors alone. The Qi’s study showed that systemic inflammation index was a routine, low-cost, and readily available serum biomarker indicating poor prognosis, and the nomogram containing Risk could achieve a better prognosis prediction of LS-SCLC. However, it has not been externally applied so far.For years, cumulative studies have confirmed the predictive role of clinical variables on prognosis, remarkably immune and inflammatory response, which has been considered one of the main prognostic predictors in several solid tumors –9. RecenThis study aimed to perform an external application of Qi’s prediction model, verify the predictive value of Risk, and evaluate the nomogram’s applicability in a single center with different case-mix characteristics.This retrospective observational external application cohort study was carried out in patients pathologically diagnosed as SCLC in Shanxi Cancer Hospital from January 2015 to December 2018. Inclusion criteria were similar to the original study . All patThe inflammatory variables included in Risk and relevant clinicopathological characteristics in the nomogram were collected. The calculation formulas of platelet-to-lymphocyte ratio (PLR), NLR, and SII were the same as those in the original study . The nomP-value of <0.05 was considered statistically significant. Replicate codes in the original study to construct Risk. The cutoff values of inflammatory variables were determined by the package “maxstat” of R software based on OS. The selection of optimal prognostic factors was performed by the package “glmnet” of R software. The multicollinearity among variables was assessed by variance inflation factors (VIFs). A cutoff value was adopted to dichotomize the total cohort into high-Risk and low-Risk groups. Differences in OS between Risk groups were estimated in the subgroup analysis. A P < 0.05 indicated statistical significant.Baseline characteristics in Qi’s and external application cohorts were described as counts (n) and percentages and compared using the Chi-square test. A The total score for each patient was calculated based on the weight of each factor in the original nomogram and was used to depict the receiver-operating characteristics (ROC) curves. The Discrimination of the nomogram was evaluated by Harrell’s concordance index (C-index) or the area under the receiver-operating characteristics (AUROC) curves. The value of AUC ranged from 0.5 (random chance) to 1.0 (perfect discrimination), and higher AUC indicated higher prediction accuracy. Then the calibration curve was plotted to visually assess the calibration of the nomogram by comparing the predicted survival probabilities with the observed survival probabilities. A close to 45-degree calibration indicated a perfect prediction model.All statistical analyses in this article were performed using R statistical software (version 3.6.3).9/L (range = 41-717 ×109/L), 1.77× 109/L (range = 0.59-4.02 × 109/L), 42.5 × 109g/L (range = 30-51.9 × 109g/L), 2.52 (range = 0.7-22.68),159.99 (range = 18.14-590.16), and 686.49 (range = 57.69-8074.23), respectively. The median age was 60 years (range 28-76 years). The median follow-up time was 20.57 months, far less than the time in the original study (55.9 months). One hundred fifty-six deaths  had been observed during follow-up, similar to the original cohort (60.8%). The median OS was 22.2 months, less than the Qi’s cohort (25.7). The 1-, 2-, 3-year OS rates were 76.1%, 45.6% and 36.2%, respectively. P=0.005) and smokers . The percentages of both the T and N stages also had statistical differences(P<0.001). Overall, our patients tend to have more advanced T and N stages. The treatment methods varied the most. Our patients received less PCI  and significantly more sequential chemoradiotherapy  than Qi’s cohort.This study included 254 LS-SCLC patients based on the inclusion and exclusion criteria. The median values of platelet, lymphocyte, albumin, NLR, PLR, and SII were 264 × 10P>0.05), as shown in Due to the different laboratory instruments used in the two hospitals, the range of reference values may be different. Varying the optimal cutoff value of each inflammatory variable is required. We copied the methods in the original article to determine the optimal cutoff values of these inflammatory markers, as shown in P<0.05).Compared with the high-Risk group, patients in the low-Risk group had significantly better prognoses. The low-Risk group had a 1-, 2- and 3-year OS of 82.0%, 51.9%, and 40.6%, while the high-Risk group had a 1-,2-,3- year OS of 55.4%, 23.9%, and 20.5%, respectively. Within the study period, a total of 156 deaths occurred, 111(59.4%) in the low-Risk group and 45(78.9%) in the high-Risk group. The subgroup analysis confirmed the good prognostic capacity of Risk Figure 2We assessed the predictive performance of the nomogram in terms of discrimination and calibration. ROC-curve analysis showed that the AUCs of 1-, 2-, and 3-year OS rates were 0.644, 0.666, and 0.635 Figure 3In this study, we have conducted an external application of the Qi’s nomogram to verify the predictive value of Risk and assess the nomogram’s broad applicability. The results showed that Risk could be used as an independent predictor to distinguish prognosis in LS-SCLC patients. The nomogram showed a moderate-to-good performance in another hospital center with different inpatient characteristics.Several studies have highlighted the value of system immune-inflammation and nutritional parameters in the prognosis prediction for SCLC patients , 11–13. We found significant differences in clinicopathological features and treatment methods when comparing the baseline characteristics between the external application cohort and the Qi’s cohort. The median OS of patients in our study was shorter than that described in the Qi cohort (22.2 vs. 25.7month). The proportion of receiving PCI in our cohort was significantly lower than that in Qi’s cohort. Treatment choices in the two centers varied dramatically, with almost the opposite ratio. There were also between-group differences in other variables, such as gender, smoke, T stage, and N stage. Various factors, including the economic level, medical resources, public health policies, social habits, and environmental risks, may cause the differences. However, this is the inherent character of real-world population data.A great deal of research is being devoted to developing new prediction models , 14, 15,Data obtained in our study indicate a moderate-to-good predictive ability despite the differences mentioned earlier between cohorts of patients. Due to the short follow-up time (20.6months), we evaluated the performance of the nomogram by predicting 1-, 2-, and 3-year OS rates, rather than 1-, 3-, and 5-year OS rates in the original study. We assessed the predictive ability in terms of discrimination and calibration. The discrimination ability of the nomogram in the external application cohort, as measured by the AUC, showed a slight decrease compared to that in the original study. The AUC values of 1-, 2-, and 3-year OS rates were 0.644, 0.666, and 0.635 in our cohort, and the AUC values of 1-, 3-, and 5-year OS rates were 0.717, 0.735, and 0.719 in the original cohort. It’s not surprising that we observed a performance decline in this study. Compared to the original cohort, significantly different baseline characteristics and treatment methods of the external application cohort would affect the nomogram’s discriminative performance. Patients in our cohort were more often male, smokers, and had more advanced T and N stages. Our patients received less PCI and more sequential chemoradiotherapy. One controversy in the original nomogram was that sequential chemoradiotherapy was a favorable factor in the prognosis of patients. However, in our study, it was associated with poor OS. Yet now, the additional benefit of sequential chemoradiotherapy remains unclear for LS-SCLC patients. The above reasons may cause the reduced discrimination of the nomogram in our cohort. Though the discrimination of the nomogram is a little lower than that in the original study, it still had a good predictive ability. The calibration plot of the nomogram in the external application cohort showed a good correlation between the prediction and actual observation for 1-, 2-, and 3-year OS rates, with a calibration slope and intercept of 1.00 and 0.The nomogram is easily implemented in clinical practice to estimate individualized risk by entering three factors: PCI , Treatment , and Risk . Our external application study results indicated the nomogram could achieve modest discrimination and ideal calibration. It may be a readily valuable tool to predict LS-SCLC prognosis.A major limitation in this study was that the median follow-up time in our cohort was 20.6 months, shorter than the 55.9 months in the original cohort. So we did not analyze the 5-year OS prediction due to data limitations. Secondly, the strict selection criteria limited the external application cohort. Last, this was a single-center retrospective study; more external application from other centers with diverse inpatients is needed to assess the applicability of nomograms in the real world.The nomogram proposed by Qi et al. was successfully applied in a different population, as it showed clinical meaningful discrimination and accuracy. The Risk could be a strong serum marker in the prognosis prediction of LS-SCLC. Further application in other centers is needed to test the clinical utility of the nomogram.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics committee of Shanxi Provincial Cancer hospital (No. 202102). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.LW, QH, JL, JC, and SX contributed to conception and design of the study. QH, NY, BS, and LW organized the database. JC and QH performed the statistical analysis. LW wrote the first draft of the manuscript. JC and SX reviewed and edited the manuscript. JL, YL, XC, and HL validated the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by the Key Research and Development (R&D) Projects of Shanxi Province [grant number 201803D31174] and the Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bifidobacterium longum subsp. longum BCBL-583 isolated from a Korean female fecal sample showed no virulence factor or antibiotic resistance gene, suggesting human safety. In addition, this strain has oxygen and heat tolerance genes for food processing, and cholesterol reduction and mucin adhesion-related genes were also found. For in vivo evaluations, a high fat diet (HFD) mouse model was used, showing that BCBL-583 administration to the model (HFD-583) reduced the total cholesterol and LDL-cholesterol in the blood and decreased pro-inflammatory cytokines but increased anti-inflammatory cytokines, substantiating its cholesterol reduction and anti-inflammation activities. Subsequent microbiome analysis of the fecal samples from the HFD mouse model revealed that BCBL-583 administration changed the composition of gut microbiota. After 9 weeks feeding of bifidobacteria, Firmicutes, Actinobacteria, and Bacteroidetes increased, but Proteobacteria maintained in the HFD mouse models. Further comparative species-level compositional analysis revealed the inhibitions of cholesterol reduction-related Eubacterium coprostanoligenes and obesity-related Lactococcus by the supplementation of B. longum BCBL-583, suggesting its possible cholesterol reduction and anti-obesity activities. The correlation analysis of HFD-583 between the gut microbiota compositional change and cholesterol/immune response showed that Verrucomicrobia, Firmicutes, Actinobacteria, and Bacteroidetes may play an important role in cholesterol reduction and anti-inflammation. However, correlation analysis of Proteobacteria showed the reverse correlation in HFD-583. Interestingly, the correlation analysis of B. longum ATCC 15707 administration to HFD model showed similar patterns of cholesterol but different in immune response patterns. Therefore, this correlation analysis suggests that the microbial composition and inflammatory cytokine/total-cholesterol may be closely related in the administration of BCBL-583 in the HFD mice group. Consequently, BCBL-583 could be a good probiotic strain for gut health promotion through gut microbiota modulation.Complete genome sequence analysis of  Bifidobacterium is one of the dominant colonizers in the human gastrointestinal tract (GIT). It is well-known that bifidobacteria may have various potential benefits for health promotion such as alleviation of diarrhea and constipation, immune regulation, anti-tumorigenesis, recovery of healthy gut microbiota after antibiotics treatment, and vitamin biosynthesis (via fructose-6-phosphate phosphoketolase (F6PPK) pathway for growth inhibition of acid-sensitive gut pathogens by lowering luminal pH  patients prevented CD4+ lymphocyte over-activation and reduced Interleukin (IL)-6 and Interferon (IFN)-γ in the serum suggesting an anti-inflammation activity (B. longum NCC2705 induced the production of monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-α by stimulation of Toll-like receptor 9 (TLR9) in RAW 264.7 macrophages , bile acid, and vitamin D . HoweverB. longum strains  from the elderly showed strain specificity in vitro, suggesting that B. longum has two different cholesterol reduction mechanisms, bile salt deconjugation by bile salt hydrolase and/or cholesterol assimilation by specific absorption as mentioned previously. To evaluate them, each B. longum strain was administrated to a high-cholesterol diet rat model which revealed in vivo cholesterol reduction from 27% to 44% supporting these mechanisms  fed mice to evaluate the effect of the immune response . And then, this strain was selected from 753 Bifidobacterium strains in bifidobacteria culture collection and tested for the development of a new probiotic strain  L-cysteine hydrochloride (MRSc) at 37°C for 24 h under anaerobic condition. Culture media and all chemical reagents were purchased from Difco (United States) and Sigma-Aldrich (United States).c strain . Bifidobg for 5 min after cultivation. The genomic DNA was extracted and purified using the G-Spin Total DNA Extraction Kit , according to the manufacturer’s protocol. The genome of BCBL-583 was completely sequenced using PacBio RS II  by Macrogen, Inc. (South Korea). The qualified raw reads were assembled using canu-2.0 with the default parameters . Total RNA for each condition was isolated using the RiboPure RNA Purification Kit (Thermo Fisher Scientific) following the manufacture’s protocol. The mRNA expression levels were analyzed with the One Step TB Green® PrimeScript™ RT-PCR Kit II  on the CFX96 system . The sequences of the primers are listed in TC) was determined automatically using the CFX Manager™ Softerware version 3.1 (Bio-Rad). All samples were analyzed in triplicate. Expression levels were normalized, and the Real Time (RT)-PCR data were analyzed by the 2CT−ΔΔ method . After cultivation for 18 h at 37°C, the cells were collected and treated with RNAT method .Bifidobacterium longum BCBL-583 was cultivated in MRSc medium with or without 0.03% Cholesterol-Water Soluble powder (Sigma-Aldrich) to determine the cholesterol content in the medium and membrane/cytoplasm of the bacterial cell after incubation at 37°C for 18 h. The cell was harvested by centrifugation at 10,000 × g for 2 min, and the cholesterol content of supernatant was estimated. The harvested cells were resuspended with 2 ml of distilled water followed by sonication using EpiShear Probe Sonicator  in an ice bath to obtain cytoplasm. The cellular membrane was isolated according to the method described by B. longum ATCC 15707), and HFD-583 group (HFD with MRSc medium and B. longum BCBL-583). The added amount of specific strain was 108 CFU/kg/day in the group, and total feeding period was 8 weeks  attached to 1 ml syringe. For the gavage, two Bifidobacterium strains were cultivated until an optical density of 1.0 at 600 nm wavelength and then administered to the mice in each group every day. The mice body weight was measured twice a week until the end of this study.Male C57BL/6 mice (5-week-old) were obtained from RaonBio (South Korea) and acclimatized to laboratory conditions consisting of a 12:12-h light–dark cycle, 24°C, and 55% humidity for 1 week with a normal diet of D12450B , according to the manufacturer’s instructions.After 8 weeks of the experimental diets, the mice were subjected to fasting for 12 h and then sacrificed. Blood samples were collected from the heart, centrifuged at 3,000 × g for 5 min at 4°C. The pellet was resuspended to 1 ml of RPMI-1640 . After cell counting, the resuspended spleen cells were inoculated into fresh RPMI-1640 medium and incubated at 37°C for 72 h with aeration supplemented with 5% CO2. The cultivated cells were harvested using a sterilized cell scraper, and the supernatant was separated by centrifugation at 3,000 × g. The supernatant was used for estimating the cytokine levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and IL-4 by ELISA kit .To determine the cytokine levels in the spleen, it was excised after the mice was euthanized. The excised spleen was soaked in 3 ml of ice-cold Hank’s Balanced Salt Solution (HBSS) buffer (Thermo Fisher Scientific) for 1 h. It was poured onto a clean plate and chopped with a sterilized scalpel. Chopped tissue in HBSS buffer was passed through 70 mash cell strainer , and then, 10 ml of ammonium chloride solution  was added to the pass through. After incubation at 4°C for 10 min to lyse the red blood cells, a pellet was obtained by centrifugation in 300 × g, respectively. The supernatant was obtained, and the manufacturer’s instructions were followed to extract the stool DNA. All extracted total fecal DNA was quantified with the NanoDrop 2000 (Thermo Fisher Scientific) and diluted to 5 ng/μl to amplify the 16S rRNA gene by PCR.Stool samples from each mouse were collected at the first days of Week 1, Week 5, and Week 9 and stored at −80°C. Before the extraction of the stool DNA using QIAamp DNA Stool Mini Kit , the stool samples were soaked in 1.4 ml of ASL lysis buffer with stainless beads  and boiled for 10 min. After boiling, the mixtures were vortexed vigorously and centrifuged for 1 min at 10,000 × The diluted total fecal DNA was used for PCR amplification with universal primers targeting from V3 to V4 regions of the 16S rRNA gene. For amplification of the 16S rRNA, the 341_F (CCTACGGGNGGCWGCAG) and 805_R (GACTACHVGGGTATCTAATCC) universal primer set with sample-specific barcodes was used as in a previous study . The ampThe qualified sequence reads were processed with the following steps: (i) the paired end fastq sequence set of each sample was aligned by the “fastq-join” method using the default parameters ; (ii) mut-test. IBM SPSS software version 25 (United States) was used to perform all statistical tests. A value of p < 0.05 was considered statistically significant.All data are expressed as the mean ± SD. Statistical significance was assessed by Duncan’s multiple range test and Student’s B. longum BCBL-583 has a circular chromosomal DNA of 2,393,285 bp with no plasmid (attL and attR sequences (BCBL_0084 to BCBL_0095) including phage tyrosine-type recombinase and integrase but no reverse transcriptase and phage structural proteins, suggesting that it may be fragmented. In addition, VFDB and CARD analyses of this genome showed that no genes encoding toxins, virulence factors, or complete antibiotic resistance genes were detected in this genome, suggesting the safety of BCBL-583 for human applications.The complete genome sequence of  plasmid . The GC  plasmid . In totaThe genome annotation revealed that this strain might have specific functions such as complex carbohydrate degradation, cell adhesion, bacteriocin production, and cholesterol reduction . This getad) locus (BCBL_0122 to BCBL_0128) was detected in the genome, probably involved in the construction and localization of a pilus  has a role for the secretion of putative pilin proteins in the tad locus (BCBL_0126 and 0127) or FimA prepilin (BCBL_1977) using sortase A (BCBL_1976).To colonize on the mucus layer in the gut environment for survival, cell adhesion activity may be important for bifidobacteria. This genome has cell adhesion-associated protein secretion systems and encodes extracellular proteins related to mucus layer anchoring. Therefore, extracellular proteins containing a signal peptide sequence and the LPXTG motif are secreted from the cell membrane by a specific extracellular protein secretion system [signal recognition particle (SRP)-dependent proteins and Sec-dependent secretion system] and anchoring-process and maturation enzymes , a lantibiotic prepeptide (BCBL_1798), a lantibiotic response regulator (BCBL_1797), lantibiotic modification enzymes (BCBL_1796 and BCBL_1795), a lantibiotic immunity protein (BCBL_1794), and a transporter with a protease domain for the secretion of the peptide  for the Clp gene cluster  and thioredoxin systems  to convert reactive oxygen species (ROS) to H2O . In addibsh; BCBL_1011), probably associated with deconjugation and precipitation of bile salt. Due to the removal of bile salt, intestinal cholesterol should be converted to bile salt to maintain a constant level, resulting in cholesterol reduction. To further understand cholesterol reduction by BCBL-583, the systematic response to cholesterol needs was studied in vitro.It has been known that bifidobacteria may lower the cholesterol concentration . Interesbsh gene in BCBL-583 were determined using qRT-PCR with different concentrations of bile acid. A low concentration of bile acid (0.075%) increased the bsh gene transcription, but a high concentration (0.15% and 0.3%) decreased the RNA expression, suggesting that mRNA expression of the bsh gene may not be bile acid concentration-dependent, even though a low concentration of bile acid stimulated the mRNA expression  like in previous reports  compensation for a lowered concentration of bile acid after deconjugation in blood by bioconversion of cholesterol and (2) absorption and accumulation of intestinal cholesterol in the bifidobacterial cells. To clarify the first hypothesis, transcription profiles of the pression . Based o reports . Therefohe cells . HoweverB. longum BCBL-583, the levels of total cholesterol, LDL-cholesterol, and HDL-cholesterol in the blood samples of the HFD-583 mouse group were determined. The normal diet (ND) and HFD groups had a cholesterol level of 0.0014% and 0.0228%  based on the unweighted UniFrac distance  were significantly reduced in the B. longum-fed groups (HFD-15705 and HFD-583), suggesting that B. longum may have an inhibition activity against these genera bacteria  have relatively proportional correlations with total cholesterol in both HFD groups, but they may not increase the total cholesterol level probably because the administration of B. longum inhibits their growth like in the previous compositional change analyses  was found for regulation of the Hsp100 chaperon family genes (Clp gene cluster). The consensus motif sequence of CGCT-N4-GCCNA was identified upstream of the clpP1 gene to regulate expression  revealed a bacteriocin gene cluster consisting of three parts: a two-component regulatory system (lanR2K), a bisin-encoding gene (lanA), and a bisin processing gene cluster  are constituently expressed, but the bisin-encoding gene and bisin processing gene cluster are expressed only on an agar medium condition , Jih1 , and HN001 , supporting this result.Since the first bacteriocin-like compound bifidin from  in 1984 , several in 1984 . However in 1984 . These ct strain . Howeverondition . This spondition . Notablyin vivo, administration of bifidobacteria to a specific mouse model and its subsequent functional properties need to be verified. For this study, two major research objectives have to be accomplished: (1) cholesterol reduction in vivo by bifidobacteria and (2) immune regulation from obesity in vivo by bifidobacteria. To test these two major functionalities of bifidobacteria, a specific mouse model needs to be developed. In this study, a mouse model was developed using an HFD containing low carbohydrates, high lipids and high cholesterol  for the transportation of triglycerides to peripheral tissues. After hydrolysis of the triglycerides by lipoprotein lipase in the muscle and adipose tissues, the VLDL particles are reduced to low-density lipoprotein (LDL) particles. In the cells of the peripheral tissue, cholesterol uptake from LDL is used as a cell membrane component and as solubilizers of lipid. The excess cholesterol is transferred to the liver in the form of high-density lipoproteins (HDL) for reverse cholesterol transport. In the liver, cholesterol is reused to generate bile acid secreted into the intestine  and to perform the direct feeding of this bacterium to obese/lean mouse model or human participants to clarify the role of this bacterium for correlation with human health in the near future.To further understand the effect of h lipids . Therefod Week 9 . Howevers result . Howeverse model . Based oomarkers . Howeveromarkers  and thisB. longum BCBL-583 was performed in genus as well as species level. It has been known that E. coprostanoligenes has cholesterol reduction activity by bioconversion of cholesterol to coprostanol via indirect cholesterol reduction pathway. This pathway consists of oxidation (to 5-cholesten-3-one), isomerization (to cholestenone), and reduction steps , consistent with the result in this study  solved this undesirable problem regarding obesity caused by the administered HFD and resulted in a reduction of blood cholesterol, inflammation and an increase of crobiota . TherefoThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Y-TK, C-HK, Y-SS, and J-HL: conceptualization. Y-TK, C-HK, HK, and J-HL: methodology. Y-TK, C-HK, J-GK, and JC: validation, formal analysis, and investigation. Y-TK, C-HK, and J-GK: data curation and visualization. Y-SS: resources. Y-TK, C-HK, J-GK, HK, and J-HL: writing—original draft. Y-TK, J-GK, HK, and J-HL: writing—review and editing. Y-SS, HK, and J-HL: supervision. J-HL: project administration and funding acquisition. All authors contributed to the article and approved the submitted version.This research was supported by the Food Research Center, Binggrae, Co., Ltd., and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Agricultural Microbiome R&D Program (no. 918022-04-HD020). And, it was also supported by the Cooperative Research Program for Agriculture Science and Technology Development (project no. PJ0158652021) and Rural Development Administration, South Korea.C-HK and Y-SS were employed by Food Research Center, Binggrae Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Acer species) and have been associated with incidences of atypical myopathy among horses in pastures. In this work, a simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method without derivatization was developed for the quantification of HGA and MCPrG in maple samples and validated according to EU guidelines. The LOQ presented here for HGA (16.4 µg/kg) is considerably lower than the lowest published LOQ (500 µg/kg). This method confirms that sycamore and box elder maple contain considerable amounts of HGA and MCPrG. In addition, the presence of the dipeptides hypoglycin B and γ-glutamyl-MCPrG in these two maple species is shown using high-resolution MS. This is the first report on the presence of these dipeptides in maple since 1973. The presence of HGB and γ-glutamyl-MCPrG could change the way we understand animal intoxication following the ingestion of maple.Hypoglycin A (HGA) and methylenecyclpropylglycine (MCPrG) are formed by some maple trees ( Acer species) such as sycamore maple (Acer pseudoplatanus) [Acer negundo) [Hypoglycin A  and its homologue methylenecyclpropylglycine (MCPrG) are naturally occurring non-proteinogenic toxic amino acids . They arlatanus) ,8,9,10 anegundo) . Being tnegundo) ,13 and Jnegundo) ,16,17,18negundo) ,21,22,23negundo) ,25 and Bnegundo) . Due to negundo) ,28. In anegundo)  possiblyHGA and MCPrG are not toxic per se, but they are bioactivated in humans through the metabolism into the coenzyme A (CoA) adducts of methylenecyclopropylacetic acid (MCPA) and methylenecyclopropylformic acid (MCPF), respectively. MCPA-CoA and MCPF-CoA bind to the multiple acyl-CoA dehydrogenases that are necessary for the metabolism of short- and medium-chain fatty acids and branched-chain amino acids. This leads to the inability to metabolize fatty acids and the accumulation of fat esters that damage the muscle cell membrane, triggering the symptoms of intoxication ,30,31. IAcer pseudoplatanus [Other toxins such as hypoglycin B  and γ-glutamyl-α-(methylenecyclopropyl)glycine (γ-glutamyl-MCPrG) have been also reported to exist in the seeds of ackee fruits. They are the dipeptides of glutamic acid and HGA and MCPrG, respectively. HGB and γ-glutamyl-MCPrG show lower hypoglycemic activity than HGA and MCPrG but are still associated with Jamaican vomiting sickness ,34,35. Fplatanus ,37. The Several LC-MS methods for the quantification of HGA and MCPrG in different plant materials and HGB in ackee have been reported. The majority of these methods involve pre-column derivatization with o-phthalaldehyde ,39, phenAcer species was developed and applied for the screening of maple leaves, seeds and seedlings for the presence and levels of these compounds. In addition, the presence of the dipeptides HGB and γ-glutamyl-MCPrG in two Acer species was confirmed using high-resolution (HR) MS/MS. The confirmation methodology included matching HR fragmentation data with available standards/databases and systematic structure elucidation using the mzLogic algorithm and in silico fragmentation. This is the first report on the presence of HGB and γ-glutamyl-MCPrG in maple since the work of Fowden and Pratt in 1973 [In this work, a simple, sensitive and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method without derivatization for the quantification of HGA and MCPrG in  in 1973 .Acer species (A. pseudoplatanus (sycamore maple), A. negundo (box elder maple), A. campestre (field maple), A. platanoides (Norway maple), A. tataricum (Tatar maple) and A. cappadocicum (Cappadocian maple)) were screened for the presence of HGA and MCPrG using HRMS. HGA and MCPrG were not detected in A. campestre, A. platanoides, A. tataricum and A. cappadocicum. On the other hand, A. pseudoplatanus and A. negundo showed distinct signals for the toxins. Therefore, method development and validation were performed using the seeds of A. platanoides as a blank matrix. The validated method was finally applied for the quantification of HGA and MCPrG in A. pseudoplatanus and A. negundo samples.Seeds, leaves and seedlings from different A. pseudoplatanus seeds. The extraction efficiency using water and acidified water was noticeably better than using methanol. The extraction with water was preferred due to the higher extraction efficiency. The first two extraction fractions proved to contain more than 99% of the extracted HGA (The number of extraction cycles (up to three cycles) was investigated using three solvents  on the naturally contaminated cted HGA . TherefoIn this work, a simple LC-MS/MS method without derivatization was developed, optimized and validated for the quantification of HGA and MCPrG in maple samples. The MRM-extracted ion chromatograms of HGA and MCPrG are shown in The matrix effect was 48% and 53% for HGA and MCPrG, respectively. No matrix effect was observed when the extract was 1 to 25 diluted with 5% MeOH/water before LC-MS/MS measurement . The dilAcer species in Germany, only the samples of sycamore maple (A. pseudoplatanus) and box elder maple (A. negundo) showed measurable quantities of HGA and MCPrG (A. pseudoplatanus (native to Europe) and A. negundo (native to North America) are the only source of HGA among the investigated Acer species. The ingestion of the seeds and seedlings of A. pseudoplatanus is therefore associated with incidences of AM among horses on European pastures [Of the collected samples from different nd MCPrG . The datnd MCPrG  and withnd MCPrG  could bend MCPrG . These fpastures ,42. The pastures ,21. Grazpastures , while tpastures ,25, it ipastures  and cow’pastures . In ordem/z values for those peaks were identified . In addition, systematic structure elucidation using HRMS/MS data was conducted using the mzLogic algorithm, which utilizes elemental composition to search structural databases in addition to extensive high-resolution fragmentation information from mzCloud and then ranks potential structural candidates according to spectral similarity and sub-structural information. Running mzLogic resulted in HGB as the top ranked candidate  [HGB and γ-glutamyl-MCPrG are known to be present in the seeds of ripe ackee fruit. It was demonstrated that an inverse relationship exists between the levels of HGA and HGB in the seeds during the ripening process of ackee fruit. During ripening, the amount of HGA decreases while HGB level increases. This formation of HGB from HGA is believed to be mediated by the enzyme γ-glutamyl-transpeptidase  in the biosynthetic pathway . HGB is e ackee) .To the best of our knowledge, this is the first report on the presence of HGB and γ-glutamyl-MCPrG in maple in the past 50 years. In 1973, Fowden and Pratt detected HGB and γ-glutamyl-MCPrG in sycamore seeds . Since tSo far, the involvement of HGB in the development of atypical myopathy in horses has not been considered. This may be due to a lack of information on the presence and concentrations of HGB in seeds and seedlings as well as serum and/or urine samples. Further investigations are needed to understand the role of HGB in the process of poisoning and to draw possible conclusions to metabolic involvement. It is still unclear whether or to which extent HGB might transform in the gastro-intestinal tracts (GITs) or livers of farm animals. Upon ingestion by grazing animals, the hydrolysis of HGB into HGA cannot be excluded. The presence of HGB and γ-glutamyl-MCPrG could thus change the way we understand animal intoxication following the ingestion of maple. Therefore, more attention should be paid to HGB and γ-glutamyl-MCPrG, whose quantification should be integrated in the current methods of the analysis of HGA and MCPrG.Acer species was developed and applied for the screening of maple leaves, seeds and seedlings for the presence and levels of these compounds. The method included a simple extraction procedure and UPLC-MS/MS analysis without derivatization. The method was validated according to the EU guidelines and demonstrated good recovery and precision for diluted and undiluted samples. The dilution approach has the advantage of eliminating the matrix effect. The sensitivity of the developed method (an LOQ of 16.4 µg/kg for HGA) proved to be better than for already published methods (500 µg/kg for HGA). In addition to the quantification of HGA and MCPrG in sycamore and box elder maple samples, the presence of the dipeptides HGB and γ-glutamyl-MCPrG in these two Acer species was confirmed using high-resolution (HR) MS/MS.In summary, a simple, sensitive and validated method for the quantification of HGA and MCPrG in The method proposed in this work allows for the quick and sensitive quantification of HGA and MCPrG in maple samples. It can be also adapted to include the quantification of HGB and γ-glutamyl-MCPrG once reference standards are available. In addition to the detection of noticeable levels of HGA and MCPrG in sycamore and box elder maple samples, the HRMS/MS analyses confirm the presence of the dipeptides HGB and γ-glutamyl-MCPrG in maple at levels that are probably higher than those of HGA and MCPrG, respectively. These dipeptides have been overlooked in maple for decades. Further investigations are needed to examine the relevance and impact of the presence of HGB and γ-glutamyl-MCPrG on the course of intoxications as well as the potential risks to humans.4COOH) were purchased from Merck . Double-deionized water was obtained using a Milli-Q system from Merck .(S)-hypoglycin A  and α-(methylenecyclopropyl)glycine (MCPrG) standards were purchased from Toronto Research Chemicals . Acetonitrile (ACN), methanol (MeOH), formic acid (FA) and ammonium formate (NHAcer species (A. pseudoplatanus (sycamore maple), A. negundo (box elder maple), A. campestre (field maple), A. platanoides (Norway maple), A. tataricum (Tatar maple) and A. cappadocicum (Cappadocian maple)) were collected from different sites in the Berlin-Brandenburg region in the north-east of Germany and North Rhine-Westphalia in the western part of Germany. The samples were air-dried and then homogenized using knife/ball mills . The homogenized material was stored in a dry place at room temperature.Seeds, leaves and seedlings/young shoots from different Five mL deionized water was added to 0.5 g homogenized plant material. The mixture was vortexed and then sonicated in an ultrasonic bath  for 10 min  at room temperature. Subsequently, the sample was centrifuged for 10 min at 4000 rpm  at room temperature. The supernatant was then filtered  into a new 15 mL Falcon tube. The residues of the extracted sample were extracted again with another 5 mL water as above, centrifuged, filtered and combined with the first extract supernatant. Samples (undiluted or 1 to 25 diluted with 5% methanol/water) were then measured by LC-MS/MS.v/v). A working standard mixture (1.0 µg/mL) was prepared by mixing stock solutions and dilution with 5% MeOH in water (v/v). The mixture served both as a calibration mixture and as a spiking solution for validation. For calibration, a series of solutions at 0.5, 1, 2, 3, 4, 5, 10, 25, 50 and 100 ng/mL HGA and MCPrG were prepared in 5% MeOH and blank extract .Stock HGA and MCPrG standard solutions (0.1 mg/mL) were prepared in 50% ACN in water , coupled to a Q-Trap 6500+ mass spectrometer  equipped with an IonDrive™ Turbo V electrospray ionization (ESI) source. Chromatographic reversed-phase (RP) separation with 10 μL injection volume was achieved on a Waters Acquity UPLC BEH C18 column  with guard column  at a flow rate of 0.3 mL/min and a column oven temperature of 40 °C. The binary mobile phase consisted of 5 mM ammonium formate and 0.1% FA in water (eluent A) and methanol (eluent B). The gradient elution was adopted as follows: 0 min 0% B; 1 min 0% B; 2 min 50% B; 3 min 70% B; 5 min 100% B; 7 min 100% B; 7.5 min 0% B; 10 min 0% B. MS detection was conducted using positive electrospray ionization (ESI) and measuring in multiple reaction monitoring (MRM) mode with mass transitions and MS conditions shown in m/z with a resolution of 70,000 and automatic gain control (AGC) setting of 3 × 106 with a maximum injection time (IT) of 100 ms. For confirmation, data-dependent MS2 (dd-MS2) was applied. In dd-MS2, the most abundant precursor ions in each full scan were selected by the quadrupole and then sent to the higher-energy collisional dissociation (HCD) cell for ion fragmentation and finally to the Orbitrap mass analyzer for detection. The dd-MS2 was performed at a mass resolution of 17,500, intensity threshold of 6.0e4, isolation width of 1.0 m/z and normalized collision energy (NCE) of 35% with ±20% step. This method was used for the screening of different Acer species samples for the presence of HGA and MCPrG (full scan mode) and for the structure elucidation of HGB and γ-glutamyl-MCPrG (dd-MS2).The samples were analyzed using an UltiMate 3000 UPLC coupled to a QExactive Focus mass spectrometer  equipped with a heated electrospray ionization (HESI) source. Chromatographic reversed-phase (RP) separation was performed using the same conditions mentioned above. HRMS was performed in positive ionization mode. The HESI temperature was set at 400 °C, the capillary temperature at 256 °C, the electrospray voltage at 3.5 kV and S-Lens RF level at 60. Sheath and auxiliary gas flow rates were 47.5 and 11.25 L/min, respectively. All data in this study were acquired using a full scan mode covering the mass range from 80 to 500 Acer species were screened for the presence of HGA and MCPrG using HRMS. Seeds of Acer platanoides (Norway maple) were used as a blank matrix for method validation. The method was validated according to the European Union SANTE/2019/12682 guidelines [Samples from different idelines . The metLinearity and range: a series of matrix-matched standard solutions in the range of 0.5–100 ng/mL HGA and MCPrG were evaluated. Deviation of back-calculated concentration from true concentration should be ≤±20%.Limit of detection (LOD) and limit of quantification (LOQ): LOD and LOQ were estimated according to the EURL Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food  using spRecovery: four quality control (QC) samples were prepared by spiking and then extracting blank samples. The QC levels were: LOQ (20 µg/kg), low , medium  and high . The average recovery for each QC level should be within 70–120%.r) and within-laboratory reproducibility  were estimated for the QC samples. RSDr and RSDwR for each QC level should be ≤20%.Precision: repeatability . LC-HRMS/MS data evaluation and structure elucidation were performed with Xcalibur, ver. 4.4 and Mass Frontier, ver. 8.0 SR1 software .


Major depressive disorder (MDD) refers to a mental disease with complex pathogenesis and treatment mechanism. S-ketamine exhibited high effectiveness in treating MDD. However, the pharmacological activity of S-ketamine has not been reported yet.In this study, depression-like characteristics were induced by chronic unpredictable stress (CUS). After S-ketamine (15 mg/kg) was intraperitoneally injected, the behaviors of mice were tested by conducting open-field test, elevated plus maze test, tail suspension test, and forced swimming test. Bilateral injection of sirtuin type 1 (SIRT1) inhibitor EX-527 was injected into the medial prefrontal cortex (mPFC) to upregulate the SIRT1 expression. The expression of SIRT1 and brain-derived neurotrophic factor (BDNF) was detected by conducting Western blot and immunofluorescence assays. Meanwhile, the synaptic ultrastructure was detected by transmission electron microscopy.In this study, the mice showed depression-like behavior in a series of behavioral tests. After the treatment with S-ketamine, the depression-like behavior stopped. Further, the synaptic ultrastructure in mPFC, including the decreased curvature of the post synaptic density and thinning of the postsynaptic density, improved after the S-ketamine treatment. Moreover, we found that S-ketamine had the possibility of spontaneous binding with SIRT1 at the molecular level and reversed CUS-induced SIRT1 reduction. Meanwhile, a positive relationship between SIRT1 and BDNF expression in mPFC and SIRT1 inhibitor limited the role of S-ketamine in reducing the depression-like behavior and increasing the BDNF level.S-ketamine upregulated the SIRT1-mediated BDNF in mPFC and reversed the synaptic structural defects caused by CUS. SIRT1 is a mediator of S-ketamine in alleviating depression-like behavior. Major depressive disorder (MDD) refers to a frequently occurring and recurrent psychiatric disorder characterized by low mood, anhedonia, thoughts of worthlessness, inappropriate sense of guilt, and cognitive disturbances. More than 350 million people worldwide suffer from MDD, accounting for about 4.4% of the global population . CurrentThe level of brain-derived neurotrophic factor (BDNF), a neurotrophic protein, remarkably decreases in patients with MDD, which is considered as a risk factor for depression , 7. DeyaSirtuin type 1 (SIRT1) has been proved to be involved in many pathological mechanisms of depression, including glial activation, neurogenesis, circadian rhythm, inflammatory reaction and BDNF signaling , 13. AndTaken together, we hypothesized that SIRT1 was a mediator of S-ketamine in increasing the BDNF level, and S-ketamine alleviated depression symptoms through the SIRT1-dependent BDNF pathway. Therefore, mice exhibited depression-like characteristics due to chronic unpredictable stress (CUS). The antidepressant behavior was observed by conducting behavior tests. The SIRT1 and BDNF protein levels were measured by conducting biochemical tests in the mPFC of mice administered with S-ketamine.n = 100, 6 weeks old, 20 ± 2 g). The mice were housed, five mice per cage, in a room with a 12-h light, 12-h dark cycle (lights on at 09:00 h) at constant ambient temperature (23 ± 1°C) and relative humidity (45%). Food and water were available ad libitum. The body weight of mice was recorded daily. After 7 days of keeping mice under these conditions, the behavior of all the mice was tested. All the studies were approved by the Ethical Committee of the Cheeloo College of Medicine.The animal center at Shandong University provided C57BL/6N male mice  was obtained from Jiangsu Hengrui Medicine Co., Ltd. . Based on previous research  were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. And based on our preliminary tests, 0.5 μg EX-527 were choose to inhibit the expression of SIRT1 in mPFC.S-ketamine hydrochloride : the mice received no treatment and remained in their cages for 21 days; CUS group (n = 10): the mice were exposed to CUS for 21 consecutive days to induce depression-like behavior as reported in previous studies; Sal group (n = 10): After exposure to CUS for 21 days, the mice were treated with Sal  for a single pass; S-ket group (n = 10): After exposure to CUS for 21 days, the mice were treated with S-ketamine  for a single pass. After the treatment, all the mice were subjected to behavioral and biochemical tests. The experimental procedure is shown in The 40 mice used in this study were randomly divided into the following four groups to determine whether ketamine alleviated depression-like behavior by regulating SIRT1 and BDNF: control (n = 10): The mice received no treatment and remained in their cages for 21 days; CUS group (n = 10): The mice were exposed to CUS for 21 consecutive days to induce depression-like behavior as reported in previous studies; Sal group (n = 10): After exposure to CUS for 21 days, the mice were treated with Sal  for a single pass; S-Ket group (n = 10): After exposure to CUS for 21 days, the mice were treated with S-ketamine  for a single pass; EX-527 group (n = 10): After exposure to CUS for 21 days, the mice were treated with SIRT1 inhibitor EX-527  for a single pass; EX-527 + S-Ket (n = 10): After exposure to CUS for 21 days, the mice were treated with EX-527  and S-ketamine  for a single pass. After the treatment, all the mice were subjected to behavioral and biochemical tests. The experimental procedure is shown in This study further evaluated whether SIRT1 was a mediator of S-ketamine in increasing BDNF level and alleviating depression-like behavior. The 60 mice used in this study were randomly divided into the following six groups: control  to inhibit the expression of SIRT1 protein. Briefly, the mice were anesthetized with isoflurane and placed in a stereotaxic frame. Next, 0.5 μg EX-527 was injected bilaterally into the mPFC   of mice The open-field test (OFT) was used for assessing anxiety-like behavior in a CUS model in previous study . The opeThe elevated plus maze test (EPM) was used to assess anxiety-like behavior as described previously . After tThe tail suspension test (TST) can reliably assess depression-like behavior. In this study, the mouse was suspended upside down by tails above the floor (60 cm) using adhesive tape placed at the height of 1 cm from the tip of the tail. The time of immobility and the motion heat map were recorded for 6 min using a video camera . The criterion for immobility was defined as the body being completely motionless and hanging passively.The forced swimming test (FST) was conducted to determine depression-like behavior in mice. In this study, the mice were placed in a clear glass cylinder (25 cm high and 10 cm in diameter) filled with water of 25 cm height (24 ± 1°C). The time of immobility and the motion heat map were recorded for 6 min using a video camera . The criteria for immobility were defined that the animal is moving at a speed less than 40 cm/s.The mice were quickly decapitated, and the prefrontal lobe was separated. Subsequently, the brain tissue was homogenized using lysis buffer  and centrifuged at 12,000 rpm at 4°C for 15 min. The supernatants were reserved, and the protein samples were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then transferred to a polyvinylidene fluoride  membrane using a Trans-Blot wet transfer system . After incubating with an antigen-blocking solution for 2 h, a PVDF membrane was sequentially incubated with BDNF , SIRT1 , PSD95 , and β-actin  antibody for overnight. After washing with phosphate buffer saline (PBS) for three times, the PVDF membrane was incubated with HRP-labeled Goat Anti-Mouse IgG (H + L)  and HRP-labeled Goat Anti-Rabbit IgG (H + L)  for 2 h. An image was obtained after ECL  development by the Amersham Imager 680 . And the band intensities were normalized to β-actin.After perfusion with PBS and 4% paraformaldehyde , the mouse brain was fixed with paraformaldehyde for 48 h. Subsequently, coronal cryotome sections (20 μm) were cut through the mPFC using a cryostat and washed with PBS three times. After incubating with 0.5% Triton X-100  for 30 min and an antigen-blocking solution at ambient temperature for 1 h, the brain sections were sequentially incubated with BDNF  and SIRT1  antibody for overnight. Next, after washing with PBS for three times, the brain sections were incubated with Alexa Fluor 488-labeled Goat Anti- Rabbit IgG (H + L)  and Alexa Fluor 555-labeled Donkey Anti-Mouse IgG (H + L) . The nucleus was counterstained with DAPI . Ten randomly selected non-overlapping fields of x400 magnification were collected from three mPFC tissue per group. An image was obtained using a ZEISS microscope . And the numbers of SIRT1–BDNF–co-labeled cell were automatically measured using ZEISS Imaging Elements Software .The mice were perfused with 30 mL of 0.1 mol/L PBS, followed by perfusion with 50 mL of fixative of an electron microscope specimen . Next, the brain tissue was decapitated at a low temperature, and the mPFC was bluntly dissected. The mPFC tissue (about 1 mm × 1 mm × 1 mm) was separated and fixed with 4% glutaraldehyde for 2 h and 1% osmium tetroxide for 2 h. The tissue was then embedded with epoxy resin. The hypothalamus sample was cut into slices, about 50 nm thick, and soaked in uranium dioxane acetate for 45 min for electron staining. Neuronal ultrastructure images were collected using an H-7650 transmission electron microscope. Ten synapses form three PFC tissue per group were selected for ultrastructure analysis accordance with the Guldner and Jones’ methods , 25.1. The structures of S-ketamine (Compound CID: 182137) were obtained from PubChem2. PyMOL (V 2.4.0) was used to dehydrate the protein, delete the original ligand, and hydrogenate and calculate the charge. The substrate ketamine was docked into the active site of protein using the AutoDock Vina tool (V1.1.2) in Chimera. The docked poses with the highest docking scores were used for further analysis. The docking result was visualized using PyMOL software.Molecular docking is widely used to predict ligand–target interactions at a molecular level . The init-test was used. P < 0.05 indicated significant statistical differences. All the data were expressed as mean ± standard error using GraphPad Prism 5.0.All the data reported in this study were obtained by an independent investigator blinded to the experimental conditions and analyzed using SPSS . The outlier  would be deleted by SPSS. A one-way ANOVA followed by Dunnett’s test were used where all groups are compared to control. When comparing the Sal group with the S-ket group and S-ket group with the EX-527 + S-Ket group, the dfbetweengroups). Meanwhile, the PSD was downregulated in the CUS group [ANOVA: dfbetweengroups) in mice , 29. In Sirtuin type 1, identified as one of the two genome-wide significant loci that contribute to depression , has nowSIRT1 participates in depression through various mechanisms, covering inflammation , BDNF siSynaptic structures can respond adaptively to changing environments, and the structural remodeling of neurons occurs after stress, participating within cognitive deficits and depression , 41. TheThis study evaluated a new mechanism by which ketamine ameliorated depression-like behavior. We found that S-ketamine upregulated the SIRT1 and BDNF expression in mPFC. Meanwhile, the use of S-ketamine reversed synaptic structural defects caused by CUS. Moreover, SIRT1 was a mediator of S-ketamine in alleviating depression-like behavior. This study provided more insights into the action mechanism of ketamine and might help identify new drug targets.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Ethical Committee of the Cheeloo College of Medicine.LH, JM, and HM developed the concept of the manuscript. LH wrote the first draft of the manuscript. XL, DW, and CL were responsible for data and manuscript review. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of  Mutations in the c-KIT gene, receptor tyrosine kinase expressed on mast cells, have been reported in tumor cells of feline MCT5, and this is believed to be one of the causes of feline MCT. A variety of c-KIT mutation patterns have been reported; however, information on c-KIT mutations in feline MCTs remains little. Since insertional mutations in exon 8 are the most frequently observed6, only a limited number of exons, mainly exons 8 and 9, have been investigated. Furthermore, whether individual mutations that have been previously reported are truly linked to c-KIT activation or tumor cell proliferation is unclear.Feline mast cell tumor (MCT), which is the most common tumor of the spleen, is a frequently encountered tumor in cats, especially in the skinc-KIT gene are found in mastocytosis and gastrointestinal stromal tumors (GISTs) in humans and in 20–30% of canine MCT6. Activating mutations in the c-KIT leads to MAPK activation7, which in turn causes tumor cell growth. Consequently, molecularly targeted drugs inhibiting c-KIT activation have been successfully used as therapeutic agents not only in GIST in humans8 but also feline MCT6. Therefore, identifying key information about c-KIT mutations and evaluating their role in the subsequent pathogenesis are very important. Several reports showed that the mutations of feline c-KIT9, and exons 8, 11, and 12 of feline c-KIT are often tested with commercial laboratory tests, whereas mutations in other sites are generally not examined because their contributions are perceived as less significant. Hence, a broad analysis of c-KIT mutations in feline MCT is very important for obtaining clinical information. In addition, it is also known that intratumor heterogeneity may be associated with tumor cell growth and selection of some tumor cells by treatment10.Mutations in the c-KIT from a cat with splenic MCT. By analyzing the nucleotide sequences of these clones, we identified the existence of multiple c-KIT clones, some of which were not previously reported in literature. Among them, certain mutations were missense mutations, i.e., causing the substitution of a single amino acid. To analyze the function of mutant c-KIT genes, the mutant proteins were exogenously expressed in mouse IL-3-dependent Ba/F3 cell lines, and some of them were proved to have functions of induction of c-KIT activation and cell proliferation.In this study, we isolated and cloned 10 cDNA clones of feline c-KIT was detected by a commercial laboratory test without any other mutations in exons 9 and 11.A 16-year-old spayed female sham cat was presented for a detailed examination of several skin masses. Physical examination revealed that six masses were present in the entire body skin, including one located at the root of the left ear, whereas the remaining masses were located on the trunk. The mass on the root of the left ear appeared one year ago, but other masses appeared just two months before initial administration to our hospital. Blood examination showed no abnormalities. Splenomegaly was identified by abdominal ultrasonography. The fine needle aspiration of the spleen allowed us to diagnose this cat with MCT in the spleen. One of the skin masses was punch-biopsied on the same day, and it turned out to be a high-grade MCT. Using the samples from skin biopsy and spleen, the exon 8 insertion (c.1245-1256dup) of c-KIT mutation analysis by commercial laboratory demonstrated that both tumors had the same c-KIT mutation (exon 8 insertion), as shown in the initial presentation. Following the removal of the spleen and skin tumors, the cat was under observation at the local doctor, and eventually died on day 191.According to the above diagnosis, we started the administration of toceranib (2.3 mg/kg) every other day and diphenhydramine (2.3 mg/kg) B.I.D. At recheck on day 19, all masses on the skin completely disappeared, followed by the disappearance of splenomegaly. However, on day 105, the mass on the root of the left ear grew again, and this mass was removed by surgery on day 117. Splenectomy was also performed on the same day. The histopathological findings of the ear mass and spleen revealed a high- and low-grade MCT, respectively, and the c-KIT cDNA clones were obtained from a spleen cDNA sample and were sequenced. As shown in Table c-KIT cDNA (NM_001009837.3), all these clones had the same silent mutation (c.2856G > A) in exon 21 and deletion of 12 nucleotides (c.1532_1543del) in exon 9 , and the four other kinds of plasmids having one of the Ins8, M537R, W854R, or Ins8-W854R mutations, which would help to identify the true functions of M537R and W854R mutations.Among these identified aberrant c-KIT-transduced Ba/F3 stable cell lines, we examined the effect of these mutations on cell proliferation  of fSCF stimulation.We also assessed the effect of the toceranib on cell proliferation of these Ba/F3 mutant cell lines Fig. . Feline c-KIT gene from the splenic tumor sample of a feline MCT, in addition to the common exon 8 insertional mutations. In addition, this cat had skin masses that were diagnosed as MCT with the same exon 8 mutations by a commercial laboratory test. We started the subsequent treatment with toceranib, and MCT in all of the skin sites disappeared. Furthermore, ultrasonography revealed an improvement in the splenomegaly and echogenicity of the spleen. However, the recurrence of skin MCT appeared after 100 days, and the cat died on day 191. This is consistent with the findings of Sabattini et al. reported that cats with skin and spleen MCT tended to die earlier than cases with an exclusive splenic involvement4. Exon 8 insertional mutations were detected by a commercial laboratory test in the samples both before and after the treatment, including skin mass and spleen, although other mutations, such as S477I and N506I in exon 9, tested by a commercial laboratory, were not observed. However, the range of commercial laboratory test was practically limited to duplicate mutations in exon 8 and these point mutations in exon 9, suggesting that any other mutations of c-KIT were unknown.In this report, we identified the novel mutations of the 11, especially in molecularly targeted therapy, and reflects the heterogeneous proliferation. In this study, we identified several different clones from spleen MCT. We isolated five types of independent cDNA clones (one major clone and four minor clones) from ten clones. These clones were isolated from only a small part of splenic tissues. However, had we used the different parts of splenic tissues, we could have had more types of clones. Furthermore, we obtained splenic tissue for sequence analysis after the beginning of the toceranib therapy. Therefore, we cannot exclude the possibility that molecularly targeted therapy facilitated this type of heterogeneity during treatment, which may have, in turn, led one major clone to obtain a cell growth advantage or exhibit resistance to the therapy. In dogs and cats, c-KIT mutations are often reported, but to date, there are no reports on intratumoral heterogeneity. The intratumoral heterogeneity of c-KIT mutation was reported in human GIST12, and that of p53 mutation was reported in canine histiocytic sarcoma13. Recently, next-generation sequence technology and single-cell analysis methods are becoming increasingly popular. Consequently, these technologies can provide researchers with more precise information regarding heterogeneity studies.Intratumor heterogeneity is one of the characteristic features of tumors that is often related to treatment resistancec-KIT mutations between spleen and skin tumors, even among several skin tumors, because tissue samples for RNA extraction were unavailable. The c-KIT sequences of genomic DNA were also not examined by ourselves, because they were also unavailable. The comparison of genomic DNA in tumor and normal samples would provide us with the information of whether the mutations found in our study were generated intratumorally generated, skin tumor or spleen tumor that metastasized to one another, or whether they came as stemmed from intratumoral heterogeneity as reported in multiple skin MCTs in dogs14.In this study, the cat had several skin MCTs in addition to splenic MCT. Although commercial laboratory tests showed the same mutation of exon 8, we could not compare the sequence similarity of other 15, as consistent with predominant strong phosphorylation of c-KIT in Ba/F3 cell lines having Δ9 mutations in our study. However, this strong phosphorylation did not induce additive proliferation effect as compared with Ba/F3 wild type cells, but this mutation showed relative resistance to toceranib treatment at 1 µM, though Ba/F3/WT, Ba/F3/Ins8, and Ba/F3/Ins8/Δ9 had sensitivity to toceranib at 1 µM to some extent. This indicated that Δ9 mutation had some functional effect on c-KIT activation, but need to be clarified in the future.We found several new mutations that were not previously reported. c.1532_1543del mutation in exon 9 was found in all clones, indicating that this mutation may be a genomic mutation or somatic mutation obtained by MCT at the early stages of tumorigenesis. c.1532_1543del mutation in exon 9 generated the deletion of amino acids GNSK, which corresponded to the GNNK-deletion in the juxtamembrane region of the extracellular domain, often found in human and mouse c-KIT as an alternative splicing isoform. GNNK deletion is known to be induced stronger and rapid phosphorylation of c-KIT16. E839K is located in the tyrosine kinase domain, which is quite similar to the c-kit protein of the W854R mutation. As shown in Figs. c. 2560T > C mutation was found in only one clone, and this is quite a unique mutation. This led to W854R mutation, which suppressed the c-KIT activation and cell proliferation induced by c-KIT activation. In canine and feline MCT, there have been no reports on the suppressive mutation of c-KIT activation, perhaps because researchers have not attempted to examine the mutations in exon 18 using c-kit analysis. In human MCT, the E839K type of suppressive mutation was previously reportedc-KIT mutations in the transmembrane domain have been identified, and F522C mutation in the transmembrane domain of the human c-KIT was reported in a patient with mastocytosis17. F522C mutation caused the autophosphorylation of c-KIT, which is sensitive to the treatment with imatinib; this means that this mutation is responsible for cell proliferation. In this study, M537R mutation showed no advantage of cell proliferation in the absence of any cytokine, despite the fact that it seemed to influence the increase of immature form of c-KIT without fSCF. But true function of this mutation remained unclear.We also found an M537R mutation in exon 10, where was located in the transmembrane region of the c-KIT by hydrophobicity analysis. Few c-KIT on cell proliferation was revealed, which was first report about feline c-KIT. The phosphorylation at Tyr 719 of c-KIT may not represent the true c-KIT activation related to the cell proliferation effect. Instead, analyzing the other phosphorylated sites or c-KIT activation status may be a good indicator of c-KIT activation showing the phenotypic function such as cell proliferation.In this study, the relationship of the phosphorylation at Tyr 719 of c-KIT and c-KIT-dependent cell proliferation has not been clarified, although the functional effects of each mutant of feline c-KIT mutations in feline MCT and analyzed their functions. In addition, the presence of heterogeneity in feline MCT was revealed. It is expected that more comprehensive analysis of the c-KIT gene will be conducted in the future to clarify the pathogenesis of MCT in cats.In conclusion, we identified new 18. Ba/F3 cell line, which was obtained from the Cell Resource Center for Biomedical Research , was grown in R10 complete medium (RPMI1640 supplemented with the same reagents as D10 complete medium) plus 10 ng/ml of mouse IL-3 . All cell lines were maintained at 37 °C in a humidified 5% CO2 incubator.PLAT-gp cell line was kindly provided by Dr. Toshio Kitamura  and was grown in D10 complete medium (Dulbecco's modified eagle medium (DMEM) supplemented with 4500 mg/L high glucose, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 55 μM 2-mercaptoethanol)c-KIT cDNA was amplified with primers, YTM1483 (5′-CAGGAACGTGGAACGGACCTC-3′2) and YTM1509 (5′-TCTACCCTGGAACAGGATGC-3′) by PCR. The resultant PCR products were electrophoresed in agarose gel, and the expected band was extracted, followed by second PCR with YTM1527 -YTM1528  to carry BamHI-XhoI sites on both ends. The products were digested with BamHI and XhoI and cloned into BamHI-XhoI sites of pcDNA3 plasmid vector. The nucleotide sequence analysis of each plasmid was performed by the DNA Core Facility of the Center for Gene Research, Yamaguchi University, and compared with the nucleotide sequence of feline c-kit registered in the public database (NM_001009837.3).After obtaining written consent from the owner for the use of the residual sample of the spleen, which is the surgical specimen for treatment, total RNA was isolated from the spleen sample using the ISOGEN II kit , and cDNA was synthesized using the Superscript III first-strand synthesis system for RT-PCR , according to the manufacturer's instructions. Feline To obtain retroviral expression vectors, all cDNA clones in pcDNA3 were transferred into the pMx-IP vector (kindly provided by Dr. Toshio Kitamura) with FLAG-tag sequences at C-terminus. Furthermore, to obtain the other mutant forms of feline c-kit, which were not cloned in this study, PCR-based molecular techniques were used to transduce the desired mutations. All mutations were confirmed by nucleotide sequence analysis.c-KIT vectors. Briefly, PLAT-gp cell lines were transfected with one of the c-KIT expression vectors and pCAGGS-VSVG using PEI max. After 48 h, the supernatant was collected and added to Ba/F3 cell line for infection. Finally, c-KIT -transduced Ba/F3 cell lines were selected by 4 µg/ml of puromycin.Ba/F3 cell line was retrovirally transduced with mutant feline 3 cells/well, in the presence of 10 ng/ml of mouse IL-3 or 100 ng/ml feline SCF , and cultured for 48 h. After 48 h of incubation, CCK8 reagent  was added to each well, and cells were cultured for an additional 1 h. Following this 1 h incubation, the absorbance at 450 nm was measured in the plates using the ARVO (Perkin Elmer) plate reader. The same experiments were repeated three times.For the cell proliferation assay, each Ba/F3 cell line was seeded in triplicate in 96-well plates at 5.0 × 10c-KIT, each cell line was seeded in 6-well plates at 5.0 × 105 cells/well and incubated for 3 h in the plain RPMI. After incubation, cells were stimulated with fSCF (100 ng/ml) for 5 min and harvested and lysed with NP40 lysis buffer . The resulting protein lysates were loaded on SDS–polyacrylamide gels and electrophoresed followed by blotting to polyvinylidene fluoride transfer membrane . Then, the membrane was blocked with 5% skim milk blocking buffer, followed by incubation with the primary antibody, rabbit polyclonal anti-phospho-c-KIT antibody (Tyr719) , goat polyclonal anti-c-KIT antibody , or mouse monoclonal anti-beta actin antibody (Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated antibodies, including donkey anti-rabbit IgG-HRP , goat anti-mouse IgG-HRP (Bio-Rad Laboratories) and goat anti-mouse IgG-HRP (Bio-Rad Laboratories) were used as secondary antibodies. The specific proteins were detected using the ECL reagent .To evaluate the activation status of the mutant p-value of less than 0.05 was considered significant.Each assay was performed as three technical replicates and repeated as a series of at least three independent biological replicates. All of the calculated data were compared using a two-tailed one-way factorial ANOVA test followed by multiple comparisons using the Tukey–Kramer HSD test (for comparisons of more than two groups). Calculations were performed using the JMP software, ver. 14.0 . A Supplementary Information.